1. Field of the Invention
The present invention is directed to a cDNA clone encoding a human imidazoline receptive protein, designated as an imidazoline receptor subtype-1 (abbreviated IR1), and fragments thereof. Also, the invention relates to an IR1 polypeptide encoded by the cDNA, as well as fragments containing the receptor binding site(s). The invention also relates to methods for producing such a cDNA clone, methods for expressing the IR1 protein, and uses thereof.
2. Description of Related Art
It is believed that brainstem imidazoline receptors possess binding site(s) for therapeutically relevant imidazoline compounds, such as clonidine and idazoxan. These drugs represent the first generation of ligands for the binding site(s) of imidazoline receptors. However, clonidine and idazoxan are also known to possess high affinity for xcex12-adrenergic receptors. Second generation ligands, such as moxonidine, possess somewhat improved selectivity for IR1 over xcex12-adrenergic receptors, but more selective compounds for IR1 are needed.
An imidazoline receptor clone is of particular interest because of its potential utility in identifying novel pharmaceutical agents having greater potency and/or more selectivity than currently available ligands have for imidazoline receptors. Recent technological advances permit pharmaceutical companies to use combinatorial chemistry techniques to rapidly screen a cloned receptor for ligands (drugs) binding thereto. Thus, a cloned imidazoline receptor would be of significant value to a drug discovery program.
Until now, the molecular nature of imidazoline receptors remains unknown. For instance, no amino acid sequence data for IR1, e.g., by N-terminal sequencing, has been reported. Three different techniques have been described in the literature by three different laboratories to visualize imidazoline-selective binding proteins (imidazoline receptor candidates) using gel electrophoresis. Some important consistencies have emerged from these results despite the diversity of the techniques employed. On the other hand, multiple protein bands have been identified, which suggests heterogeneity amongst imidazoline receptors. These reports are discussed below. Some of the abbreviations used hereinbelow, have the following meanings:
Reis et al. [Wang et al., Mol. Pharm., 42: 792-801 (1992);
Wang et al., Mol. Pharm., 43: 509-515 (1993)] were the first to demonstrate partial purification of an imidazoline-selective binding protein and to characterize it as having MW=70 kDa. This was accomplished using bovine cells (BAC), which lack an xcex12AR [Powis and Baker, Mol. Pharm., 29:134-141 (1986)]. The 70 kDa imidazoline-selective protein in those studies had high affinities for both idazoxan and p-aminoclonidine affinity chromatography columns. To date, no one has reported the complete purification of this imidazoline receptor protein. Likewise, no amino acid seguences have been reported for IR1.
The partially purified 70 kDa protein was used by Reis and co-workers to raise xe2x80x9cI-site binding antiserumxe2x80x9d, designated herein as Reis antiserum. The term xe2x80x9cI-sitexe2x80x9d refers to the imidazoline binding site, presumably defined within the imidazoline receptor protein. Reis antiserum was prepared by injecting the purified protein into rabbits [Wang et al, 1992]. The first immunization was done subcutaneously with the protein antigen (10 xcexcg) emulsified in an equal volume of complete Freund""s adjuvant, and the next three booster shots were given at 15-day intervals with incomplete Freund""s adjuvant. The polyclonal antiserum has been mostly characterized by immunoblotting, but radioimmunoassays (RIA) and/or conjugated assay procedures, i.e., ELISA assays, are also conceivable [see xe2x80x9cRadioimmunoassay of Gut Regulatory Peptides: Methods in Laboratory Medicine,xe2x80x9d Vol. 2, chapters 1 and 2, Praeger Scientific Press, 1982].
The present inventors and others [Escriba et al., Neurosci. Lett. 178: 81-84 (1994)] have characterized the Reis antiserum in several respects. For instance, the present inventors have discovered that human platelet immunoreactivity with Reis antiserum is mainly confined to a single protein band of MW=33 kDa, although a trace band at 85 kDa was also observed. This 33 kDa band was enriched in plasma membrane fractions as expected for an imidazoline receptor. Furthermore, the intensity of this band was found to be positively correlated with non-adrenergic 125PIC Bmax values at platelet IR1 sites in samples from the same subjects, with an almost one-to-one slope factor. In addition, the nonadrenergic 125PIC binding sites on platelets were discovered by the present inventors to have the same rank order of affinities as IR1 binding sites in brainstem [Piletz and Sletten, J.Pharm. and Exper. Therap., 267: 1493-1502 (1993)]. The platelet 33 kDa band may also be a product of a larger protein, since in human megakaryoblastoma cells, which are capable of forming platelets in tissue cultures, an 85 kDa immunoreactive band was found to predominate.
Immunoreactivity with Reis antiserum does not appear to be directed against human xcex12AR and/or MAO A/B. This is significant because xcex12AR and MAO A/B have previously been cloned and also bind to imidazolines. The present inventors have obtained selective antibodies and recombinant preparations for xcex12AR and MAO A/B, and these proteins do not correspond to the 33, 70, or 85 kDa putative IR1 bands. Thus, there is substantial evidence that, at least in human platelets, the Reis antiserum is IR1 selective.
Another antiserum was raised by Drs. Dontenwill and Bousquet in France [Greney et al., Europ. J. Pharmacol., 265: R1-R2 (1994); Greney et al., Neurochem. Int., 25: 183-191 (1994); Bennai et al., Annals NY Acad. Sci., 763: 140-148 (1995)] against polyclonal antibodies for idazoxan (designated Dontenwill antiserum). This anti-idiotypic antiserum inhibits 3H-clonidine but not 3H-rauwolscine (xcex12-selective) binding sites in the brainstem, suggesting it interacts with IR1 [Bennai et al., 1995]. As shown in FIG. 1, human RVLM (same as NRL) membrane fractions displayed bands of 41 and 44 kDa, as detected by the present inventors using this anti-idiotypic antiserum.
The present inventors have found that the bands of MW=41 and 44 kDa detected by Dontenwill antiserum may be derived from an 85 kDa precursor protein, similar to that occurring in platelet precursor cells. An 85 kDa immunoreactive protein is obtained in fresh rat brain membranes only when a cocktail of 11 protease inhibitors is used. Also, as shown in FIG. 1, it is found that Reis antiserum detects the 41 and 44 kDa bands in human brain when fewer protease inhibitors are used.
Additionally, the Dontenwill antiserum weakly detects the platelet 33 kDa band. Thus, the present inventors have hypothesized that the 41 and 44 kDa immunoreactive proteins may be alternative breakdown products of an 85 kDa protein, as opposed to the platelet 33 kDa breakdown product.
In summary, the main conclusion from the above results is that, despite vastly different origins, the Reis and Dontenwill antisera both detect identical bands in human platelets, RVLM, and hippocampus.
Using yet another technique, a photoaffinity imidazoline ligand, 125AZIPI, has also been developed to preferentially label I2-imidazoline binding sites [Lanier et al., J.Biol.Chem., 268: 16047-16051 (1993)]. The 125AZIPI photoaffinity ligand was used to visualize 55 kDa and 61 kDa binding proteins from rat liver and brain. It is believed that the 61 kDa protein is probably MAO, in agreement with other findings [Tesson et al., J.Biol.Chem., 270: 9856-9861 (1995)] showing that MAO proteins bind certain imidazoline compounds. The different molecular weights between these bands and those studied by the present inventors is one of many pieces of evidence that distinguishes IR1 from I2 sites.
To the inventors"" knowledge and as described herein, we are first to clone a cDNA encoding a protein with the immunological and ligand binding properties expected of an IR1. We are first to identify the nucleotide sequence of a DNA molecule encoding an imidazoline receptor, and first to determine the amino acid sequence of an imidazoline receptor. The polypeptides described herein are clearly distinct from xcex12AR or MAO A/B proteins.
The present invention is for an isolated polypeptide that is receptive to (binds to) an imidazoline compound. Exemplary imidazoline compounds in this context are p-iodoclonidine and moxonidine. Typically, such a polypeptide immunoreacts with Reis antiserum and/or Dontenwill antiserum.
In one aspect of the invention, a polypeptide includes a 651 amino acid sequence as shown in SEQ ID No. 6. Other imidazoline receptive proteins homologous to this polypeptide are also contemplated. Such a protein generally has a molecular weight of about 60 to 80 kDa. More particularly, it can have a molecular weight of about 70 kDa.
In another aspect of the invention, a polypeptide includes a 390 amino acid sequence as shown in SEQ ID No. 7. Such a polypeptide generally has a molecular weight of about 35 to 45 kDa. More particularly, it can have a molecular weight of about 37 kDa.
A DNA molecule encoding an aforementioned imidazoline-receptive polypeptide is also contemplated. Such a DNA molecule, e.g., a cDNA derived from mRNA, can contain a nucleotide sequence encoding the amino acid sequence shown in SEQ ID No. 6. Thus, a DNA molecule containing the 1678 base pair (b.p.) (1677/3=559) nucleotide sequence shown in SEQ ID No. 2 is contemplated. In another embodiment, a DNA molecule includes the longer nucleotide sequence shown in SEQ ID No. 3.
In another embodiment of the invention, a DNA molecule contains a nucleic acid sequence encoding the amino acid sequence shown in SEQ ID No. 7. Such a DNA molecule can include the 895 base pair nucleic acid sequence shown in SEQ ID No. 4. In another aspect, it can include the 1171 base pair nucleic acid sequence shown in SEQ ID No. 5.
RNA molecules complementary to an instant DNA molecule, e.g., an mRNA molecule (sense) or a complementary cRNA molecule (antisense), is a further aspect of the invention.
A further aspect of the invention is for a recombinant vector, as well as a host cell transfected with the recombinant vector, wherein the recombinant vector contains at least one of the nucleotide sequences shown in SEQ ID Nos. 2-5, or a nucleotide sequence homologous thereto.
A method of producing an imidazoline receptor protein is another aspect of the invention. Such a method entails transfecting a host cell with an aforementioned vector, and culturing the transfected host cell in a culture medium to generate the imidazoline receptor.
A significant further aspect of the invention is a method of screening for a ligand that binds to an imidazoline receptor. Such a method can comprise culturing an above-mentioned host cell in a culture medium to express imidazoline receptor proteins, followed by contacting the proteins with a labelled ligand for the imidazoline receptor under conditions effective to bind the labelled ligand thereto. The imidazoline receptor proteins can then be contacted with a candidate ligand, and any displacement of the labelled ligand from the proteins can be detected. Displacement of labelled ligand signifies that the candidate ligand is a ligand for the imidazoline receptor. These steps can be performed on intact host cells, or on proteins isolated from the cell membranes of the host cells.